BtGal35A

Ex-Gal0063

(EC.3.2.1.23)exo-β-1,4-Galactosidase

CAZy Family: GH35

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~79 kDa)

Figure 1. Electrophoresis analysis of BtGal35A. M, molecular weight marker (PageRuler Prestained Protein Ladder, Thermo Scientific); lane 1, culture lysate before IPTG induction; lane 2, culture lysate after IPTG induction; lane 3, BtGal35A purified from Ni sepharose fastflow column.

2.SPECIFIC ACTIVITY

0.29 U/mg protein (on pNP-β-Gal) at pH 7.0 and 37°C.

One Unit of pNP-β-Gal activity is defined as the amount of enzyme required to release 1 μmol of p-nitrophenyl per minute from pNP-β-Gal (5 mM) in phosphate buffer (20 mM) pH 7.0.

3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1. Relative activity of BtGal35A on different substrates^a.

^aReactions were performed with 5mM substrate, pH 7.0, at 37°C for 10 min.

^bAbsorption caused by released p-nitrophenol was measured at 405 nm. The relative activity on pNP-β-Gal was taken as 100%.

^cThe data are reported as means±standard errors from the mean for three independent experiments.

Table 2.Kinetics ofBtGal35A

Figure 2. Analysis of the influence of galactosidase activity. The galactooligosaccharides at 1 mM, were incubated with 1 μM of enzyme for 16 h using standard conditions and the reactions were analysed by HPAEC. The data presented are representative of biological replicates n = 4.

4.PHYSICOCHEMICAL PROPERTIES

pH Optima: 7.5

Temperature Optima:37°C

5.STORAGE CONDITIONS

The enzyme should be stored at -20°C. For assay, this enzyme should be diluted in phosphate buffer (20 mM) pH 7.0. Swirl to mix the enzyme immediately prior to use.

6. REFERENCES

[1] Luis AS, Briggs J, Zhang X, et al. Dietary pectic glycans are degraded by coordinated enzyme pathways in human colonic Bacteroides. Nat Microbiol. 2018 Feb;3(2):210-219.